Jesús M. Ureña

A Role for a PDZ Protein in the Early Secretory Pathway for the Targeting of proTGF-α to the Cell Surface

In general, plasma membrane integral proteins, such as the membrane-anchored growth factor proTGF-alpha, are assumed to be transported to the cell surface via a nonregulated, constitutive pathway. proTGF-alpha C-terminal mutants are retained in an early secretory compartment. Here, using a two-hybrid screen, we identify two TACIPs (proTGF-alpha cytoplasmic domain-interacting proteins) that contain PDZ domains and do not interact with proTGF-alpha C-terminal mutants. The binding specificity of one of them, TACIP18 (previously identified and named Syntenin or mda-9), coincides with that of the component that possibly mediates the normal trafficking of proTGF-alpha. TACIP18 colocalizes and interacts specifically with immature, intracellular forms of proTGF-alpha. Therefore, it appears that the interaction of TACIP18 with proTGF-alpha in the early secretory pathway is necessary for the targeting of the latter to the cell surface.

Regulation of Nogo and Nogo receptor during the development of the entorhino-hippocampal pathway and after adult hippocampal lesions.

Axonal regeneration in the adult CNS is limited by the presence of several inhibitory proteins associated with myelin. Nogo-A, a myelin-associated inhibitor, is responsible for axonal outgrowth inhibition in vivo and in vitro. Here we study the onset and maturation of Nogo-A and Nogo receptor in the entorhino-hippocampal formation of developing and adult mice. We also provide evidence that Nogo-A does not inhibit embryonic hippocampal neurons, in contrast to other cell types such as cerebellar granule cells. Our results also show that Nogo and Nogo receptor mRNA are expressed in the adult by both principal and local-circuit hippocampal neurons, and that after lesion, Nogo-A is also transiently expressed by a subset of reactive astrocytes. Furthermore, we analyzed their regulation after kainic acid (KA) treatment and in response to the transection of the entorhino-hippocampal connection. We found that Nogo-A and Nogo receptor are differentially regulated after kainic acid or perforant pathway lesions. Lastly, we show that the regenerative potential of lesioned entorhino-hippocampal organotypic slice co-cultures is increased after blockage of Nogo-A with two IN-1 blocking antibodies. In conclusion, our results show that Nogo and its receptor might play key roles during development of hippocampal connections and that they are implicated in neuronal plasticity in the adult.

MAP1B Is Required for Netrin 1 Signaling in Neuronal Migration and Axonal Guidance

BACKGROUND The signaling cascades governing neuronal migration and axonal guidance link extracellular signals to cytoskeletal components. MAP1B is a neuron-specific microtubule-associated protein implicated in the crosstalk between microtubules and actin filaments. RESULTS Here we show that Netrin 1 regulates, both in vivo and in vitro, mode I MAP1B phosphorylation, which controls MAP1B activity, in a signaling pathway that depends essentially on the kinases GSK3 and CDK5. We also show that map1B-deficient neurons from the lower rhombic lip and other brain regions have reduced chemoattractive responses to Netrin 1 in vitro. Furthermore, map1B mutant mice have severe abnormalities, similar to those described in netrin 1-deficient mice, in axonal tracts and in the pontine nuclei. CONCLUSIONS These data indicate that MAP1B phosphorylation is controlled by Netrin 1 and that the lack of MAP1B impairs Netrin 1-mediated chemoattraction in vitro and in vivo. Thus, MAP1B may be a downstream effector in the Netrin 1-signaling pathway.

Nogo-A expression in the human hippocampus in normal aging and in Alzheimer disease

Myelin-associated proteins are involved in the formation and stabilization of myelin sheaths. In addition, they prevent axon regeneration and plasticity in the adult brain. Recent evidence suggests that the expression of certain myelin-associated proteins (e.g. Nogo-A) can be regulated by synaptic activity or by over-expression after neural lesions in brain syndromes such as temporal lobe epilepsy. However, no studies on Alzheimer disease (AD) have been reported in which cell loss and significant synaptic reorganization occurs. In the present study, we analyze in detail the expression of Nogo-A in the hippocampal formation in normal human aging and in AD. Our results indicate that Nogo-A is expressed by oligodendrocytes and neurons in the aged hippocampal formation. In addition, both granule cells and mossy fiber connections are also labeled in the old-aged hippocampi. Interestingly, Nogo-A is over-expressed by hippocampal neurons in AD and is associated with &bgr;-amyloid deposits in senile plaques. Taken together, our results reinforce the hypothesis that Reticulon proteins such as Nogo-A participate in the neuronal responses stemming from hippocampal formation during senescence, and particularly in AD. These findings also indicate that Reticulon proteins could be considered as new putative drug targets in therapies of neurodegenerative disorders.

A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

Directed cell migration and axonal guidance are essential steps in neural development. Both processes are controlled by specific guidance cues that activate the signaling cascades that ultimately control cytoskeletal dynamics. Another essential step in migration and axonal guidance is the regulation of plasmalemma turnover and exocytosis in leading edges and growth cones. However, the cross talk mechanisms linking guidance receptors and membrane exocytosis are not understood. Netrin-1 is a chemoattractive cue required for the formation of commissural pathways. Here, we show that the Netrin-1 receptor deleted in colorectal cancer (DCC) forms a protein complex with the t-SNARE (target SNARE) protein Syntaxin-1 (Sytx1). This interaction is Netrin-1 dependent both in vitro and in vivo, and requires specific Sytx1 and DCC domains. Blockade of Sytx1 function by using botulinum toxins abolished Netrin-1-dependent chemoattraction of axons in mouse neuronal cultures. Similar loss-of-function experiments in the chicken spinal cord in vivo using dominant-negative Sytx1 constructs or RNAi led to defects in commissural axon pathfinding reminiscent to those described in Netrin-1 and DCC loss-of-function models. We also show that Netrin-1 elicits exocytosis at growth cones in a Sytx1-dependent manner. Moreover, we demonstrate that the Sytx1/DCC complex associates with the v-SNARE (vesicle SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and that knockdown of TI-VAMP in the commissural pathway in the spinal cord results in aberrant axonal guidance phenotypes. Our data provide evidence of a new signaling mechanism that couples chemotropic Netrin-1/DCC axonal guidance and Sytx1/TI-VAMP SNARE proteins regulating membrane turnover and exocytosis.

Bcl-2 overexpression delays caspase-3 activation and rescues cerebellar degeneration in prion-deficient mice that overexpress amino-terminally truncated prion

Prnp knockout mice that overexpress an amino‐truncated form of PrPc (ΔPrP) are ataxic and display cerebellar cell loss and premature death. Studies on the molecular and intracellular events that trigger cell death in these mutants may contribute to elucidate the functions of PrPc and to the design of treatments for prion disease. Here we examined the effects of Bcl‐2 overexpression in neurons on the development of the neurological syndrome and cere‐bellar pathology of ΔPrP. We show that ΔPrP overexpression activates the stress‐associated kinases ERK1–2 in reactive astroglia, p38 and the phosphorylation of p53, which leads to the death of cerebellar neurons in mutant mice. We found that the expression of ΔPrP in cell lines expressing very low levels of PrPc strongly induces the activation of apoptotic pathways, thereby leading to caspase‐3 activation and cell death, which can be prevented by coexpressing Bcl‐2. Finally, we corroborate in vivo that neuronal‐directed Bcl‐2 overexpression in ΔPrP mice (ΔPrP Bcl‐2) markedly reduces caspase‐3 activation, glial activation, and neuronal cell death in cerebellum by improving locomotor deficits and life expectancy.—Nicolas, O., Gavín, R., Braun, N., Ureña, J. M., Fontana, X., Soriano, E., Aguzzi, A., del Río, J. A. Bcl‐2 overexpression delays caspase‐3 activation and rescues cerebellar degeneration in prion‐deficient mice that overexpress amino‐terminally trun‐cated prion. FASEB J. 21, 3107–3117 (2007)

PrP(106-126) activates neuronal intracellular kinases and Egr1 synthesis through activation of NADPH-oxidase independently of PrPc.

Prion diseases are characterised by severe neural lesions linked to the presence of an abnormal protease‐resistant isoform of cellular prion protein (PrPc). The peptide PrP(106–126) is widely used as a model of neurotoxicity in prion diseases. Here, we examine in detail the intracellular signalling cascades induced by PrP(106–126) in cortical neurons and the participation of PrPc. We show that PrP(106–126) induces the activation of subsets of intracellular kinases (e.g., ERK1/2), early growth response 1 synthesis and induces caspase‐3 activity, all of which are mediated by nicotinamide adenine dinucleotide phosphate hydrogen‐oxidase activity and oxidative stress. However, cells lacking PrPc are similarly affected after peptide exposure, and this questions the involvement of PrPc in these effects.

Role of Sp1 in the Induction of p27 Gene Expression in Vascular Smooth Muscle Cells In Vitro and After Balloon Angioplasty

Abstract —The abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an important role in atherosclerosis and restenosis. Although several studies have implicated the growth inhibitory protein p27Kip1 (p27) in the control of myocyte growth and hypertrophy, little is known about the molecular mechanisms that regulate p27 expression in the cardiovascular system. In the present study, we demonstrate the interaction of the transcription factor Sp1 with 2 GC-rich sequences within the p27 promoter in cultured VSMCs. Importantly, point mutations that disrupted Sp1 binding markedly reduced p27 promoter activity, demonstrating that Sp1 is required for efficient p27 gene transcription in cultured VSMCs. Because p27 expression is upregulated after balloon angioplasty, we investigated Sp1 expression and activity in control and balloon-injured rat carotid arteries to assess the role of Sp1 as a physiological regulator of p27 expression. Although immunohistochemical analysis disclosed Sp1 protein expression in both control and balloon-injured arteries, a high level of Sp1 DNA-binding activity was found only in response to balloon angioplasty. Collectively, these results demonstrate that Sp1 is essential for maximum p27 promoter activity in VSMCs and suggest that posttranslational induction of Sp1 DNA-binding activity contributes to the induction of p27 expression and VSMC growth arrest at late time points after balloon angioplasty.

The CREB/CREM Transcription Factors Negatively Regulate Early Synaptogenesis and Spontaneous Network Activity

The family of CREB (cAMP response element-binding protein) transcription factors are involved in a variety of biological processes including the development and plasticity of the nervous system. In the maturing and adult brain, CREB genes are required for activity-dependent processes, including synaptogenesis, refinement of connections and long-term potentiation. Here, we use CREB1NescreCREM−/− (cAMP-responsive element modulator) mutants to investigate the role of these genes in stimulus-independent patterns of neural activity at early stages. We show that lack of CREB/CREM genes specifically in neural tissue leads to increased synaptogenesis and to a dramatic increase in the levels of spontaneous network activity at embryonic stages. Thus, the functions of CREB/CREM genes in neural activity differ in distinct periods of neural development.

Expression, synaptic localization, and developmental regulation of Ack1/Pyk1, a cytoplasmic tyrosine kinase highly expressed in the developing and adult brain

Cytosolic tyrosine kinases play a critical role both in neural development and in adult brain function and plasticity. Here we isolated a cDNA with high homology to human Ack1 and mouse Tnk2. This cDNA directs the expression of a 125‐kD protein that can be autophosphorylated in tyrosines. Initially, this clone was named Pyk1 for proline‐rich tyrosine kinase (Lev et al., 1995 ); however, since it corresponds to the mouse homolog of Ack1, here we called it Ack1/Pyk1. In this study we show that Ack1/Pyk1 mRNA and protein is highly expressed in the developing and adult brain. The highest levels of Ack1/Pyk1 expression were detected in the hippocampus, neocortex, and cerebellum. Electron microscopy studies showed that Ack1/Pyk1 protein is expressed in these regions both at dendritic spines and presynaptic axon terminals, indicating a role in synaptic function. Furthermore, we demonstrate that Ack1/Pyk1 mRNA levels are strongly upregulated by increased neural activity, produced by intraperitoneal kainate injections. During development, Ack1/Pyk1 was also expressed in the proliferative ventricular zones and in postmitotic maturing neurons. In neuronal cultures, Ack1/Pyk1 was detected in developing dendrites and axons, including dendritic tips and growth cones. Moreover, Ack1/Pyk1 colocalized with Cdc42 GTPase in neuronal cultures and coimmunoprecipitated with Cdc42 in HEK 293T cells. Altogether, our findings indicate that Ack1/Pyk1 tyrosine kinase may be involved both in adult synaptic function and plasticity and in brain development. J. Comp. Neurol. 490:119–132, 2005.